HIV/AIDS is a significant health risk to both male and female veterans, and is transmitted predominantly by heterosexual intercourse. A roadblock to the discovery of efficacious prevention modalities, including vaccines and microbicides, is the incomplete understanding of basic virus-host interactions that underlie the acquisition of mucosal HIV-1 infection in the female genital tract. Two recent NIH workshops on Mucosal Immunity and HIV Prevention highlighted under-explored fundamental questions that need to be addressed, including anatomical sites of early virus infection in the female reproductive tract (FRT), and the influence of endogenous sex hormones on the mucosal environment and susceptibility to HIV-1. We submit that the endometrial mucosa of the uterus constitutes a site of early HIV-1 infection. (i) The upper FRT (uFRT) is accessible to pathogens. The uterus is readily accessible to sperm, experimental microspheres, viral and bacterial pathogens, and possibly HIV-1 virions attached to sperm. (ii) The cervical mucus is an imperfect barrier and upward myometrium contractions likely promote delivery of semen content into the uFRT. (iii) The uFRT comprises an abundance of HIV-1 target cells beneath the epithelium lining. (iv) Cellular and innate immune functions in the uFRT are suppressed during the secretory phase of the menstrual cycle. (v) Previous studies on HIV-1 infection of UEC are. (vi) Innovative and transformative virologic strategies are available in our laboratory that will facilitate physiologically relevant studies using primary mucosal transmission models, including transmitted/founder (T/F) HIV-1 infectious molecular clones (IMC), and HIV- 1 reporter genomes that augment both sensitivity and specificity for dissecting early virus-host mucosal interactions. (vii) We present preliminary results that to our knowledge demonstrate for the first time UEC are susceptible to infection by T/F. Therefore, there exists essential virologic underpinnings and compelling rationale to further examine the uFRT as a potential site for the acquisition of mucosal HIV-1 infection and transmission. Our central hypothesis is that the uFRT mucosa is vulnerable to productive T/F HIV-1 infection. To test this hypothesis, we propose the following specific aims: (1) To determine the susceptibility of primary UEC to productive infection by T/F, chronic control and laboratory strains of HIV-1. Viruses expressing T/F envs from isogenic reporter proviral genomes will be studied, enabling sensitive and specific analyses of productive HIV-1 infection in a primary UEC model of virus infection. Importantly, chronic control viruses will be analyzed in parallel experiments to rigorously address the question of whether T/F viruses possess unique fitness properties for infecting potential target cells that exist in the uFRT mucosa. (2) To elucidate cis and trans infection pathways by which T/F HIV-1 spreads from primary UEC to endometrial CD4+ T lymphocytes. Mucosal CD4+ T cells isolated from endometrium will first be analyzed for their susceptibility to infection by T/F and control viruses to establish important underpinnings for subsequent experiments with UEC. The following experiments will elucidate susceptibilities of autologous CD4+ T cells to HIV-1 infection mediated by de novo produced virus from infected UEC or transcytosis of intact/infectious virus particles. The results will be analyzed to elucidate virus subtype- and strain-specific susceptibilities of UEC and CD4+ T lymphocytes to HIV-1 infection, and identify mechanisms/pathways of virus entry and correlations of these factors with menstrual cycle stage. Such findings promise to provide critical underpinnings for translational prevention research initiatives.